Members of the myosin family of actin filament based molecular motors perform a wide range of critical cellular functions including cellular and organelle motility, tension generation, signal transduction and transcriptional regulation. This project will explore in vivo functions of myosins in the intestinal epithelial cell (enterocyte) with particular focus on Myo9b, a myosin implicated in inflammatory bowel disease (IBD), and on the functional synergy between Myo6, the sole minus-end motor linked to the IEC brush border membrane (BBM), and Myo1a, the principal BBM-associated plus-end motor. These studies will utilize available mouse mutants for each of these myosins, as well as a Myo1a/Myo6 double mutant (DM). Myo9b contains a GTPase activating protein (GAP) domain for Rho in its tail. It is likely that Myo9b-linked IBD is due to hyperactivation of Rho, a critical regulator of the actin cytoskeleton. In vitro studies have implicated Myo9b in enterocyte monolayer wound healing and maintaining tight junction (TJ) integrity and paracellular barrier properties. Preliminary studies indicate that Myo9b knock out (KO) mice under unstressed conditions exhibit hallmark symptoms of IBD including presence of erythrocytes in the gut lumen, greatly elevated enterocyte cell death and apoptosis, inter-enterocyte invasion of immune response cells and altered junctional properties. Taken together these observations indicate that the Myo9b KO should be a powerful and novel animal model for IBD, and because these symptoms occur in both small and large intestine, for Crohn's disease in particular. Proposed studies will provide a thorough phenotypic characterization of this IBD model, including effects on enterocyte polarity, cytoskeletal organization, and of particular importance to IBD, TJ composition/organization. The mucosal barrier properties of the Myo9b KO will be quantified and the predicted hyper-sensitive response to mucosal inflammation and injury will be assessed. Studies to investigate the functional synergy between Myo1a and Myo6 will include pilot studies to test the hypothesis that Myo1a suppresses the tumor promoting activity of Myo6 through sequestration of this putative proto-oncogene to the BB. Other studies will quantify the relative contributions of Myo6 versus Myo1a to the tethering of the BBM to the actin cytoskeleton, and to the in vivo generation and shedding of BBM luminal vesicles that may be critical for protection from bacterial pathogens. Another set of studies will investigate the molecular bases for potential restoration of WT BBM function (e.g. Ca2+ transport, lipid raft organization, BBM micro-viscosity, CFTR Cl- channel activity) when both Myo1a and Myo6 are absent. Finally the synergistic roles of Myo1a and Myo6 endocytic and exocytic trafficking of BBM proteins (e.g. cystic fibrosis transmembrane conductance regulator, Na+/phosphate exchanger 2b,Toll-like receptor 4) will be explored.